| In eukaryotic cells, phosphatidylcholine (PC) is one of the most essential components. as the main component of the membrane structure, and it’s also participate in a number of physiological activity of cells, such as lipoprotein’s assembly and transport. In prokaryotic cells, phosphatidylcholine is found mainly in the symbiotic bacteria and pathogens. Previous studies have shown that, PC has been related to various interactions between prokaryotic cells,and when during prokaryotic cells infecting host cells. The primarily pathway that PC affects those bacterial secretion process is influence the factors secretion and biological activity. Effector cells are mainly secreted proteins with various kinds of biological activity. According to the bacteria pathway,it classified into the type I-VIII secretion system and the chaperone-usher(CU) secretion system. The type V secretion system(T5SS), effector proteins which belong to in secreted proteins have been proven with the most species and the most widely distributed. The type VI secretion system (T6SS) has been shown involved in a variety of processes host cells infections. Pseudomonas divided into several sub-species, widely distributed in nature, have been found to have more than one sub-species and plant and animal life and human activity is closely related to a variety of animals and plants and pathogens of human diseases. Where Pseudomonas aeruginosa is one of the most common opportunistic pathogens can cause a variety of animal wound infection, or sepsis. Thus explore the mechanism and influencing factors between Pseudomonas and target cells has important practical significance. This study aimed to explore the PC is missing in the cell membrane under conditions by Pseudomonas aeruginosa T5SS and effector protein secretion and biological activity of T6SS specific secretion is affected, for the work and made the following conclusions.(1) Effects of gene cloning, protein expression and purification. Query the database data to identify Pseudomonas aeruginosa T5SS and T6SS specific secretory protein classification and characteristics, from which to select T5SS secreted protein PlpD and EstA, VI secreted proteins Tse1and Tse3. Query of Pseudomonas aeruginosa PA01gene sequences of plpD, estA, tse1, tse3, primers were designed from Pseudomonas aeruginosa27853in the transfer of gene connected cloning vector, gene sequencing and comparison after confirmed the target gene. The target gene connected expression vector into an appropriate expression strain, the pre expression and optimize expression conditions, the successful expression of target genes. Depending on the condition expression corresponding to develop purification scheme, successful relatively pure protein, protein concentration, the amount of purified protein was calculated. When a sufficient amount of purified protein, using appropriate methods preserved.(2) Preparation of polyclonal antibodies. The purified protein antigen after treatment, many times after immunization, using enzyme-linked immunosorbent assay (ELISA) to detect rabbit polyclonal serum antibody titers. After standard, take a rabbit polyclonal antibody serum. Utilization of the polyclonal antibodies. Against P. aeruginosa ATCC27853, P. aeruginosa ATCC27853pcs-cytoplasmic protein, interstitial proteins, extracellular proteins were detected by Western blot results showed that deletion of the V-PC secreted proteins PlpD and EstA, the type VI protein secretion and Tse1/Tse3secretion showed no significant effect.(3) Comparative effects of enzyme activity, the type V protein secretion and EstA PlpD are esterase, can catalyze the decomposition of the same fatty acid substrates, but PlpD secreted into the extracellular medium, EstA attached to the cell outer membrane. Enzyme-substrate reaction system designed to compare the relative activity, the results confirmed the wild-type strain P. mutant strain P. aeruginosa ATCC27853pcs-protein PlpD no significant difference effect on enzyme activity and EstA P.aeruginosa ATCC27853and. the type VI secreted proteins Tse1and Tse3belong lysozyme protein, can be injected into the adjacent T6SS heterologous strains, inhibit or kill heterologous strains. Designing antibacterial test, and do statistical analysis of experimental data, the results confirm the enzyme activity proteins of Tse1and Tse3from wild-type strains of P. Aeruginosa and mutant strain P. aeruginosa ATCC27853pcs-no significant difference P. aeruginosa ATCC27853. |
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